There are two major challenges in the field of plant transformation:

1. Plant transformation technologies: recent developments • There are two major challenges in the field of plant transformation: • Stabilizing gene expression or Avoiding Gene Silencing • Multigene engineering or gene stacking • Issue: • Most of the transgenic plants produced by conventional methods succumb to gene silencing. • Most of the methods are suitable for transfer of one or two genes. Many traits are governed by multiple genes. Therefore, multi-gene engineering is needed to move into future biotechnology.

2. Approaches for stabilizing gene expression: • These are based on: • Designing transformation vector judiciously. • Controlling transgene integration process. • Suppress host silencing machinery • Approaches for multigene engineering or gene stacking: • Make a large transformation vector. • Integrate one by one into a predetermined genomic position. • Plastids • Minichromosomes

3. Influence of the transformation system on gene expression • Agrobacterium-mediated • Gives simpler patterns of integration. • Inserts genes in ‘expressed areas’ of the genome. • However, • Not efficient for site-specific integration • Still several complex integration loci are generated • Particle bombardment • Gives more complex integration patterns • No site preference. • Non-polar transfer. • Efficient for site-specific integration • Simpler vector system.

4. Strategies Tissue culture negatively influences transgene-expression stability • Therefore, • Limit tissue culture phase. • Limit exposure to phytohormone containing media. • Use embryogenesis, if possible, rather than organogenesis. Modifying locus structure after transformation: Multi-copy lines can be included in breeding program after converting them to single-copy lines • Conversion of complex locus to single copy was shown by different methods: • Transposition of a single-copy into a new location. • Deletion of extra copies by Cre-lox to recover single-copy

5. Solution: Monitor homozygous plants over a few generations to ensure transgene stability. Issue: Silencing may occur in homozygous plants even if their hemizygous parents were stable. Issue: Plasmid backbone integration is not only undesirable, it may also cause expression instability. Its integration may induce methylation due to the mismatch with genomic isochore. Solution: Apply backbone de-selection strategy by using negative selection markers: codA, tms2 (see below). This works well with T-DNA vectors but not with particle bombardment vectors. Integration of this vector can be selected based on NPT expression (kanamycin resistance), and cells containing backbone can be eliminated based on tms2 expression (sensitive to indole acetamide) RB- GOI- NPT-LB tms2

6. Integration site and base composition Plant genomes are mosaics of compositionally homogenous DNA segments with defined GC content, termed isochores. Because the GC content of genes of different origins, insertion of foreign DNA into an isochore may mark this region for inactivation and methylation. In this respect, modification of transgene sequences should not be limited to optimization of the codon usage to that of the host species but, ideally, should be broadened to make sure that all sequences match isochore composition. Since plant genomes consist of mosaics of isochores, matching base composition means site-specificity. This is particularly true of alloploid plants, because they contain two or more genomes. E.g.. Tobacco (allotetraploid) consists of two parental genome. Hypomethylated vs hypermethylated regions of the genome. Site-specificity can be obtained by gene targeting employing Cre-lox system. Or the transgene can be insulated with MARS to protect from surrounding problems.

7. Construct design suitable for transgene expression stability: • Promoter choice: use plant promoters and avoid using viral promoters. • Avoid inverted repeat structures or inverted transcription units (see below) • (Promoter X-Gene 1-nos3’:: nos3’-Gene 2-Promoter X or Y) • May use MARS to stabilize gene expression Matrix attachment regions (MARs) are operationally defined as DNA elements that bind specifically to the nuclear matrix in vitro. It is possible, although unproven, that they also mediate binding of chromatin (DNA + histones) to the nuclear matrix in vivo, and alter the topology of the genome in interphase nuclei. When MARs are positioned on either side of a transgene their presence usually results in higher and more stable expression in transgenic plants or cell lines, most likely by minimizing gene silencing. Our review explores current data and presents several plausible models to explain MAR effects on transgene expression. (From: Plant Mol Biol. 2000 Jun;43(2-3):361-76)

8. Expression of site-specifically integrated GUS gene in tobacco Issue: variableposition and structure of the locus cause variation in transgene expression between individual transformants containing identical gene construct Approach: Inserted GUS gene site-specifically using Cre-lox system. (for molecular strategy see lecture 10 slide 7). Result: All transgenic lines containing the site-specific integration of the GUS gene express GUS gene at more or less at the same level. No gene silencing, if viral promoter is not used. Reference: Day et al. (2000). Genes Dev. 14, 2869 2880. Chawla et al. (2006) Plant Biotech J. 4: 209 – 218

9. Using Mutant Hosts To Avoid Gene Silencing: host is impaired in the gene silencing pathway From: Plant J. 39(3):440-9, 2004 Basic and applied research involving transgenic plants often requires consistent high-level expression of transgenes. However, high inter-transformant variability of transgene expression caused by various phenomena, including gene silencing, is frequently observed. Here, we show that stable, high-level transgene expression is obtained using Arabidopsis thaliana post-transcriptional gene silencing (PTGS) sgs2 and sgs3 mutants. In populations of first generation (T1) plants transformed with a β-glucuronidase (uidA or GUS) gene driven by the 35S cauliflower mosaic virus promoter (p35S), the incidence of highly expressing transformants shifted from 20% in wild type background to 100% in sgs2 and sgs3 backgrounds. Likewise, when sgs2 mutants were transformed with a cyclin-dependent kinase inhibitor 6 gene under control of p35S, all transformants showed a clear phenotype typified by serrated leaves, whereas such phenotype was only observed in about one of five wild type transformants. p35S-driven uidA expression remained high and steady in T2 sgs2 and sgs3 transformants, in marked contrast to the variable expression patterns observed in wild type T2 populations. We further show that T-DNA constructs flanked by matrix attachment regions of the chicken lysozyme gene (chiMARs) cause a boost in GUS activity by fivefold in sgs2 and 12-fold in sgs3 plants, reaching up to 10% of the total soluble proteins, whereas no such boost is observed in the wild type background. MAR-based plant transformation vectors used in a PTGS mutant background might be of high value for efficient high-throughput screening of transgene-based phenotypes as well as for obtaining extremely high transgene expression in plants.

10. From: Plant J. 39(3):440-9, 2004 Schematic representation of T-DNA vectors pp35S-uidA, ppCASuidA, ppOMA1-uidA and pMAR-p35S-uidA. Not to scale. uidA: β-glucuronidase coding region; pat: phosphinothricin acetyltransferase coding region; pNOS: nopaline synthase promoter; p35S: cauliflower mosaic virus 35S promoter; pCAS: cassava vein mosaic virus promoter; pOMA1: hybrid octopine and mannopine synthase promoter; tOCS: octopine synthase terminator; tNOS: nopaline synthase terminator; tg7:terminator of gene 7 of Agrobacterium tumefaciens; chiMAR: chicken lysozyme MAR; RB and LB: right and left T-DNA border, respectively.

11. From: Plant J. 39(3):440-9, 2004

12. Engineered minichromosomes in plants Genetic engineering for complex or combined traits requires the simultaneous expression of multiple genes, and has been considered as the bottleneck for the next generation of genetic engineering in plants. Minichromosome technology provides one solution to the stable expression and maintenance of multiple transgenes in one genome. For example, minichromosomes can be used as a platform for efficient stacking of multiple genes for insect, bacterial and fungal resistances together with herbicide tolerance and crop quality traits. All the transgenes would reside on an independent minichromosome, not linked to any endogenous genes; thus linkage drag can be avoided. Engineered minichromosomes can be easily constructed by a telomere-mediated chromosomal truncation strategy. This approach does not rely on the cloning of centromere sequences, which are species specific,and bypasses any complications of epigenetic components for centromere specification. Thus, this technique can be easily extended to all plant species. The engineered minichromosome technology can also be used in combination with site-specific recombination systems to facilitate the stacking of multiple transgenes. From: Current Opinion in Biotechnology 2007, 18:425–431

13. Production of minichromosomes: • Radiation induced chromosomal breakage • From B chromosomes by a breakage fusion-bridge (BFB) cycle initiated in a specialized translocation between the B chromosome and the short arm of • chromosome 9 From: Current Opinion in Biotechnology 2007, 18:425–431

14. A chromosomes: Normal set of chromosomes B chromosome: super-numeray chromosomes (mostly inert) Yu W, Han F, Gao Z, Vega JM, Birchler JA: Construction and behavior of engineered minichromosomes in maize. Proc Natl Acad Sci U S A 2007, 104:8924-8929. This paper describes the targeting of minichromosomes with genes for applications in plant genetic engineering. It provides proof of concept that engineered minichromosomes can be used as platforms for the second generation technology of ‘output trait’ GM crops. Also demonstrated that maize supernumerary B chromosomes can support foreign gene expression and can be modified as vectors for genetic engineering.